Yoshifumi Takei

Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a promising molecular target to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis. Highlights Elevated METTL9 correlates with metastasis in human scirrhous gastric cancer. This is the first report on the biological relationship between METTL9 and metastasis. METTL9 protein localizes mainly in mitochondria in metastatic scirrhous gastric cancer. METTL9 knockdown reduces mitochondrial Complex I activity to decrease cell migration and invasion in metastatic scirrhous gastric cancer. METTL9 holds promise against peritoneal dissemination of scirrhous gastric cancer.

Cancer metastasis is the leading cause of death in patients with any type of solid cancer. To develop effective cancer treatments, it is essential to understand the molecular mechanisms underlying cancer metastasis. Previously we established peritoneal metastasis cell models derived from human scirrhous gastric cancer patients. In this article, we focus on the CELSR1 gene, which is involved in Wnt signaling but whose association with peritoneal metastasis is still unclear. We unveiled gene alterations and the prognostic relevance of the CELSR1 gene in cancer patients by analyzing public resources for cancer genomic and patient cohorts. RT-qPCR and immunoblot analyses revealed that CELSR1 expression was significantly elevated in our cell lines, which had high peritoneal metastatic property, compared with their parental cell lines, which had lower peritoneal metastatic property. Some of the gene alterations in the coding region of CELSR1 were observed in our metastatic cell lines, but they were not associated with the metastatic property or with patient prognosis. Knockdown of CELSR1 via the shRNA technique significantly decreased migration and invasion in the cell lines having high peritoneal metastatic property, whereas the knockdown did not significantly affect proliferation. These results show that CELSR1 plays an important role in peritoneal metastasis and that CELSR1 is a novel peritoneal metastasis-associated gene. The results also suggest that CELSR1 is a proper molecular target for therapy against peritoneal metastasis of scirrhous gastric cancers.

Objectives: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer. Methods: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination. Results: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination – and significantly prolonged the survival – in the orthotopic tumor mouse model. Conclusion: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.

The 2006 discovery of the process of RNA interference opened the door to application of this phenomenon for disease treatments. Short interfering RNA (siRNA) can be used to induce RNA interference, and this approach has generated much interest as a new type of nucleic acid-based drugs in humans and other mammals. However, despite the great potential of siRNA-based drugs in cancer therapy, some drawbacks of siRNAs, such as their instability in vivo and poor cellular uptake, remain unresolved. Here, we review the development of siRNA-based drugs targeting the human Bcl-xL gene, an anti-apoptotic factor overexpressed in many cancers, including prostate cancers. We also introduce a novel application of the biomaterial atelocollagen as a vehicle to functionally deliver tumor-specific siRNA molecules in nude mice. Here, we introduce an orthotopic tumor inoculation model in nude mice: the resulting orthotopic tumors more closely replicate the clinical conditions in humans—including the metastasis mode—than ectopic subcutaneously inoculated tumors.

Lymphatic vessels drain excess tissue fluids to maintain the interstitial environment. Lymphatic capillaries develop during the progression of tissue fibrosis in various clinical and pathological situations, such as chronic kidney disease, peritoneal injury during peritoneal dialysis, tissue inflammation, and tumor progression. The role of fibrosis-related lymphangiogenesis appears to vary based on organ specificity and etiology. Signaling via vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor (VEGFR)-3 is a central molecular mechanism for lymphangiogenesis. Transforming growth factor-β (TGF-β) is a key player in tissue fibrosis. TGF-β induces peritoneal fibrosis in association with peritoneal dialysis, and also induces peritoneal neoangiogenesis through interaction with VEGF-A. On the other hand, TGF-β has a direct inhibitory effect on lymphatic endothelial cell growth. We proposed a possible mechanism of the TGF-β–VEGF-C pathway in which TGF-β promotes VEGF-C production in tubular epithelial cells, macrophages, and mesothelial cells, leading to lymphangiogenesis in renal and peritoneal fibrosis. Connective tissue growth factor (CTGF) is also involved in fibrosis-associated renal lymphangiogenesis through interaction with VEGF-C, in part by mediating TGF-β signaling. Further clarification of the mechanism might lead to the development of new therapeutic strategies to treat fibrotic diseases.

Patients with B cell lymphomas bearing MYC translocation combined with translocation involving other genes, such as BCL2, BCL3, or BCL6, defined as double-hit lymphoma (DHL), have a poor prognosis. Recent studies expanded the concept to include double-expressing lymphoma (DEL) that co-overexpresses MYC protein with either of those proteins. Accordingly, we defined cytogenetic DHL and DEL as primary DHL. An adoptive T cell immunotherapy with a chimeric antigen receptor (CAR) has been clinically shown to exhibit cytotoxicity in refractory neoplasias. We revealed the marked cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against primary DHL cells from patients. CD19- and/or CD38-specific T cells were co-cultured with cytogenetic DHL (n = 3) or DEL (n = 2) cells from five patients for 3 days. We examined whether T cells retrovirally transduced with each vector showed cytotoxicity against DHL cells. Anti-CD19- and/or anti-CD38-CAR T cells were co-cultured with primary DHL cells at an E:T ratio of 1:2 for 3 days. Anti-CD19- and anti-CD38-CAR T cells completely abrogated these DHL cells, respectively. Anti-CD19-CAR T cells synergistically exerted collaborative cytotoxicity against these primary DHL cells with anti-CD38-CAR T cells. Therefore, refractory DHL cells can be efficiently abrogated by the clinical use of T cells with anti-CD19- and/or anti-CD38-CAR.

The mechanism and function of cancer metastasis-associated microRNAs (miRNAs) have not been completely examined. Using a miRNA array, we previously determined that a significantly decreased level of miR-516a-3p was closely associated with the peritoneal dissemination of scirrhous gastric cancers. We previously established HSC-58 cells from a scirrhous gastric cancer patient, and we further isolated metastatic cells (58As9) from ascites in nude mice upon repeated orthotopic transplantation of HSC-58. In our previous report (Takei Y, et al. Cancer Res, 2011), we showed that the miR-516a-3p expression was significantly low and the peritoneal dissemination was significantly high in 58As9 compared with HSC-58. To augment the decreased expression of miR-516a-3p in 58As9 cells, we successfully prepared a cell line (58As9-miR-516a-3p) that stably overexpressed the miRNA. In the present study, we showed that orthotopic transplantation of 58As9-miR-516a-3p into nude mice resulted in significantly decreased primary tumor growth, ascites, and peritoneal dissemination. Moreover, the transplanted nude mice showed long survival time, probably due to the small amount of ascitic fluids pooled. The miRNA directly targeted sulfatase 1, which works to remove a sulfate group from heparan sulfate proteoglycans on the cell surface, and promotes the release of membrane-bound Wnt ligands into medium. Significantly increased concentrations of Wnt3a, Wnt5a, and nuclear-accumulated b-catenin were observed in 58As9 cells, and in 58As9-miR-516a-3p, all of the levels were attenuated. Anti-metastatic therapy via injection of the miR-516a-3p expression vector/atelocollagen mixture into 58As9 orthotopic tumors in nude mice resulted in significantly prolonged survival along with the inhibition of ascites and peritoneal dissemination. Our findings thus indicate that the miR-516a-3p-sulfatase 1-Wnt b-catenin route can be targeted to block the peritoneal dissemination of scirrhous gastric cancers.

The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of amino-modified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5′- and/or 3′-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3′-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3′-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.